Genome-wide profiling of the PIWI-interacting RNA-mRNA regulatory networks in epithelial ovarian cancers

Abstract

PIWI-interacting (piRNAs), ~23-36 nucleotide-long small non-coding RNAs (sncRNAs), earlier believed to be germline-specific, have now been identified in somatic cells, including cancer cells. These sncRNAs impact critical biological processes by fine-tuning gene expression at post-transcriptional and epigenetic levels. The expression of piRNAs in ovarian cancer, the most lethal gynecologic cancer is largely uncharted. In this study, we investigated the expression of PIWILs by qRT-PCR and western blotting and then identified piRNA transcriptomes in tissues of normal ovary and two most prevalent epithelial ovarian cancer subtypes, serous and endometrioid by small RNA sequencing. We detected 219, 256 and 234 piRNAs in normal ovary, endometrioid and serous ovarian cancer samples respectively. We observed piRNAs are encoded from various genomic regions, among which introns harbor the majority of them. Surprisingly, piRNAs originated from different genomic contexts showed the varied level of conservations across vertebrates. The functional analysis of predicted targets of differentially expressed piRNAs revealed these could modulate key processes and pathways involved in ovarian oncogenesis. Our study provides the first comprehensive piRNA landscape in these samples and a useful resource for further functional studies to decipher new mechanistic views of piRNA-mediated gene regulatory networks affecting ovarian oncogenesis. The RNA-seq data is submitted to GEO database (GSE83794).

Fig 1. Expression profile of PIWIL genes and proteins in human normal ovarian and cancer tissues.

A. The relative expression of thee PIWIL mRNAs analyzed by qRT-PCR analysis. The amount of PIWIL mRNAs was normalized to the endogenous control, β-actin mRNA. The fold-change was calculated based on the ratio of the normalized values of the ENOCa and SOCa to that of normal ovary. B. Western blot of three PIWIL proteins in normal ovary, ENOCa and SOCa.

Fig 2. QC analysis of tissue samples of ENOCa, SOCa and normal ovary for small RNA sequencing and generation of reads.

Agilent RNA Bioanalyzer profile of A. ENOCa; B. SOCa; C. normal ovary; D. RIN values revealing small RNA intactness optimal for sequencing; E. Number of trimmed reads of 16–40 nts generated from each sample type.

Fig 3. Characteristic properties of piRNAs identified in ENOCa, SOCa and normal ovary.

A. Length and B. nucleotide bias observed among the piRNAs identified in each samples.

Fig 4. The chromosomal origin and genomic contexts of piRNAs in human genome.

A. Origin of piRNAs on all human chromosomes in three samples; B. piRNAs mapped to various genomic contexts of human genome.

Fig 5. The biogenetic sequence signatures of the intron derived piRNAs (piRtrons).

The preference of 1U & 10A are observed among piRtrons in ENOCa, SOCa and normal ovary.

Fig 6. The chromosomal distribution of piRNA clusters.

Location of piRNA clusters on human chromosomes in A. Normal ovary, B. ENOCa and C. SOCa samples.

Fig 7. Box plot analysis showing distribution of conservation scores of piRNAs originated from various genomic contexts in three samples (Normal ovary, ENOCa, SOCa).

Center lines show the medians; box limits indicate the 25^th and 75^th percentiles as determined by R program; whiskers extend to minimum and maximum values; crosses represent sample means; bars indicate 90% confidence intervals of the means; width of the boxes is proportional to the square root of the sample size.

Fig 8. piRNA-target duplexes in ENOCa and SOCa.

A. The binding sites of piR-52207 with its targets NUDT4, MTR, EIF2S3 and MPHOSPH8 in ENOCa; B. The binding sites of piR-33733 and piR-52207 with its targets LIAS and ACTR10, PLEKHA5 respectively in SOCa.

Fig 9. Validation of expression of piRNAs in ovarian cancer subtypes with respect to normal ovarian tissue by qRT-PCR.

Fig 10. Expression profile of piRNA targets by qRT-PCR in EOCa subtypes.

A. The relative expression of genes targeted by piR-52207 in ENOCa; B. The relative expression of genes targeted by piR-33733 and piR-52207) in SOCa.

Fig 11. The possible effects of piR-52207 on target genes and subsequent pathophysiological consequences in ENOCa.

Fig 12. The possible effects of piR-33733 and piR-52207 on target genes and subsequent pathophysiological consequences in SOCa.